New classes of therapeutic biopharmaceuticals based on peptides and proteins targeting previously non-treatable or incurable diseases have emerged in recent years, in consequence of the many new advances and developments that have arisen in the biotechnological field.
However, due to their poor oral bioavailability and general short half-lives in vivo, the delivery method of many of the proteins and polypeptide therapeutics developed thus far has been mostly restricted to the parenteral route. The human monoclonal antibodies (a rapidly growing class of targeted therapeutics) that are currently approved all require administration by injection. For example, adalimumab (Humira™, marketed by Abbott), a human monoclonal antibody first indicated for treating rheumatoid arthritis, is presented in a pre-filled syringe. Only a few oral, mucosal or inhalative therapeutic compositions are currently commercially available, many of which incorporate only relatively lower molecular weight polypeptide agents. An example of a higher molecular weight protein therapeutic delivered by inhalation route is dornase alfa (Pulmozyme™, distributed by Roche), a solution of recombinant human deoxyribonuclease I, indicated for the treatment of cystic fibrosis.
The sheer molecular size and complexity of proteins and polypeptides as well as the relative ease in loss of their activity through damage to their structural integrity poses a challenge for the processing, formulation, and delivery of these types of therapeutics.
The biological activity of a protein is dictated by its one-of-a-kind three-dimensional structure; by its secondary and tertiary structures. A specific and balanced combination of internal interactions such as hydrogen-bonding, electrostatic interactions, van der Waals forces, hydrophobic interactions, and covalent bonding between the peptide chain components encompassing the protein is what contributes to the final structure of a folded protein in its native state. Marginal changes to these interactions can potentially have a big impact on the structural integrity of a protein. The precise biological function of a protein is based on its specific interactions with other relevant macromolecules and/or small molecules. Consequently, the specificity and thus the therapeutic effectiveness of a protein will be lost, if essential three-dimensional characteristics are disrupted in any way.
The loss of native protein structure can occur through a number of degradative pathways. Aggregation can occur through non-covalent bonding events such as the self-association of native protein monomers, or the association of partially unfolded proteins into non-native oligomers. Protein deterioration and aggregation can also occur through covalent, irreversible chemical events such as the formation of and/or exchange of cross-linking disulphide bonds, peptide-bond hydrolysis, deamidation, or oxidation. The prevalence of these phenomena not only depends on the inherent characteristics of the protein, but also on a number physicochemical environmental conditions such as temperature including related stress conditions like freeze-thaw cycles, pH, protein concentration, ionic strength, the presence of destabilizing chemical additives, dryness and mechanical stress factors such as cavitation or shear, all of which can negatively impact the native folded structure of the protein.
The association of protein into such oligomeric, often high molecular weight forms poses a major problem in the formulation, delivery and long-term storage of protein or polypeptide therapeutics. Aggregation can lead to the loss of active protein drug, leading to unreliable and ineffective dosages. In liquid formulations, aggregates which may be insoluble can precipitate and form large particulates can impede flow, which would be extremely disadvantageous for parenteral applications. Furthermore, protein aggregates may exhibit toxicity and can trigger undesirable immunogenic responses (Rosenberg, A. S., AAPS J, 2006, 8, E501). The characteristics of the medium chosen for a protein or polypeptide liquid formulation can thus have a major impact towards the practicality and longevity of a formulation.
An aqueous environment is known to be important in most cases for the maintenance of protein structure and bioactivity, i.e. water molecules can be essential or even the driving force for folding and/or may play a direct role in enzymatic activity. On the other hand, an aqueous medium may also have an adverse effect, depending on the nature of the composition of the aqueous phase and various parameters such as pH and ionic strength. Water can act as a plasticizer or serve as the reaction medium as well as directly as a reaction component, such as in the hydrolytic cleavage of amide bonds. Thus, a method which has been substantially used in the field of formulating protein therapeutics is lyophilisation (freeze-drying) or spray-drying the protein to a solid-state powder form. In this case, the removal of water restricts the conformational flexibility and diffusive mobility of the protein macromolecule to interact with others, thus diminishing the chances of aggregation. Consequently, with proteins in the dry state, substantially longer-term storage is feasible, in comparison to many aqueous-based formulations.
However, it should be noted that protein degradation and aggregation can occur quite easily during the process of lyophilisation itself, and so to decrease the incidence of these events, time input and costs for developing the lyophilisation process are necessarily quite high. Additional stabilizers such as saccharides, polyols and the like, which serve to compensate the loss of water hydrogen-bonding effects are also often added into the pre-lyophilisation composition. Such excipients, while useful during the lyophilisation process may be detrimental to protein stability in the dry state over time, for instance by phase separation via crystallization. Other post-lyophilisation stabilizing excipients may also need to be included in order to support the longer shelf-life of the protein, adding to the number of components that have to be present in the final formulation.
Also, despite the removal of water, dry state protein compositions are not immune to the effects of external environmental factors such as temperature, and chemical degradation reactions where water is not a key reagent, such as deamidation or oxidation. Elevated temperatures result in increased mobility and consequently a greater likelihood for inter-protein reactions, thus many lyophilized proteins still have to be stored at all times under refrigerated conditions. Also, excipients added to render the pH and tonicity of the formulation more amenable for the lyophilisation process may not be as stabilizing for the protein in the final dry state. The introduction of moisture may be a concern, and particular attention must also be given to the storage means (as well as material) for proteins in the dry solid state.
Furthermore, the reconstitution of the lyophilized protein in aqueous media as an extra step prior to actual administration is necessary, and carries the risk of improper handling/dosing and contamination. The reconstitution step itself may trigger protein aggregation, if the pH, or temperature of the aqueous medium is not optimal or the time for proper rehydration is too short. Thus, the formulation of a suitable reconstitution medium may also need to be considered and properly developed. Overall, from an economical viewpoint, a significantly large amount of time, effort and cost are involved for the process and formulation development of lyophilized protein compared to liquid formulations (Wang, W., Int. J. Pharm., 2000, 203, 1).
The use of organic solvents as carrier media is another option for formulating protein therapeutics. It should be noted, however that such solvents may not always have a stabilizing effect on protein structure, in some cases, rather the opposite. For example, at higher concentrations, strongly polar solvents such as DMSO or DMF and alcohols such as methanol or ethanol can act as denaturants, often by competing with internal amide hydrogen-bonding which may lead to the loss of tertiary structures; even the ratio of secondary structures may be altered, possibly leading to non-native structures (Stevenson, C. L., Curr. Pharm. Biotech., 2000, 1, 165). Consequently, such solvents may not be ideal for the long-term storage of protein therapeutics. Similarly, the physiological tolerability of these types of solvents may be low, and further considerations as to the release and adsorption of the protein (also depending on the state of its solubility in such solvent systems) need to be taken into consideration.
Protein therapeutic formulations in aqueous media are often simply available in the form of a solution. Formulations using organic solvents, on the other hand, require further consideration due to the general non- or partial-solubility of the protein in such media, depending on solvent polarity and physical properties of the protein. The combination of hydrophobic organic solvents and water-free (lyophilized) protein usually produces dispersions or suspensions. In such cases, the long-term physical stability of the suspension is also an important consideration during formulation development, alongside with the long-term stability of the protein itself. The use of non-polar solvents such as oils and lipids as suspension carriers for proteins or polypeptides for parenteral use has been reported, however the stability of these carriers at physiological temperatures over extended periods of time has been questioned (Knepp, V. M. et al, Pharm. Res. 1998, 15, 1090). Further, these compounds may cause side effects as pain at injection site. Also, oils and lipids tend to strongly retard the release of therapeutic agent. This may be a useful characteristic for effecting longer term sustained release or depot-type injectable formulations, but not if a more rapid and immediate bioavailability is desired.
Polymer-based compositions have also been described, for example, viscous non-aqueous suspension formulations of protein or peptide agents, suitable for use in conjunction with an implantable device, comprising polyvinylpyrrolidone as a polymer component and lauryl lactate (or lauryl alcohol) as a solvent have been reported (U.S. Pat. No. 7,258,869 and EP1152749). Such compositions are suggested to be suited for the sustained release of such therapeutic agents.
Perfluorinated compounds have also been used as non-aqueous liquid carriers of protein, polypeptides and other biologically active agents. For example, U.S. Pat. No. 6,458,376 describes compositions proposed for ophthalmic applications (such as topically applied eye drops) in which therapeutic/diagnostic compounds, including oligopeptides and protein growth factors are suspended in perfluorocarbons and in the presence of at least one surfactant. It is however, silent on the subject of the choice of particular surfactants that can be suitable for use in compositions containing protein or peptide therapeutic compounds, and makes no discussion as to the long-term chemical and physical stability of such particular compounds in these formulations over time.
EP0939655 (and U.S. Pat. No. 6,730,328) discloses thermally stable formulations in which non-aqueous, hydrophobic, non-reactive vehicles such as mineral oil, perfluorodecalin, methoxyfluorane, perfluorotributylamine or tetradecane are used for suspension compositions comprising proteins, proteinaceous compounds and nucleic acids. The formulations are proposed for parenteral, transdermal, mucosal, oral and enteral methods of administration, as well as their use for long-term continuous administration and delivery via an implantable device. However, the ability of these suspension compositions to remain physically stable, i.e. uniformly dispersed or re-dispersible after a length of time was not disclosed. The actual tissue compatibility of these types of compositions has not been demonstrated either.
US 2010/0008996 mentions the inhalative or instillative use of SFAs as carriers for transporting an active substance to the alveolar membrane/lung regions of a patient. More in detail, the document teaches micellar colloidal solutions of active substances which exhibit sufficient solubility in SFAs and whose molecules have a size in the range of 1 to 0.1 nm in order for facilitate transport through the lung membrane to the bloodstream. It discloses SFA-based compositions of the small molecular drugs, ibuprofen, alpha-tocopherol, retinol palmitate, 5-fluorouracil, bromohexine, oseltamivir, and ambroxol, which are described as being useful for inhalation or instillation. In contrast, it does not disclose any specific composition of an active substance which is a larger molecule, such as a protein, or of an active substance which is not soluble in SFAs.
WO 2011/073134 similarly discloses solutions comprising ciclosporin, a cyclic polypeptide with molecular weight of 1202.61 in a semifluorinated alkane, optionally in the presence of a cosolvent such as ethanol. Whilst suspensions and emulsions are also mentioned as optional alternatives, there is no specific disclosure of such type of composition.
Kociok et al. (Graefe's Arch Clin Exp Ophthalmol 2005, 243, 345-358) investigated whether macrophage activation through cell membrane attachment might be supported by emulsified tamponade droplets of a certain vesicle size. For this purposes, they prepared emulsified droplets (referred to as unilamellar vesicles) of F6H8 in an aqueous continuous phase by the extrusion of the semifluorinated alkane through polycarbonate filter membranes into a PBS solution. In order to determine whether human serum albumin (HSA) has an influence on blood neutrophil activation, some of the droplets were coated with HSA by including HSA into the aqueous PBS solution.
It is an object of the present invention to introduce novel protein or polypeptide compositions which overcome the limitations and disadvantages associated with currently known formulations.